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






   
Alternate Selectivity for Polar Compounds in Hydrophilic
Interaction Liquid Chromatrography (HILIC) Using a New
Amino Type HILIC Column
Regina Roemling,1 M. Sakata,2 Y. Kawai,2 H. Yamasaki2 and H. Moriyama,2
1
Tosoh Bioscience, Stuttgart, Germany, 2Tosoh Corporation, Tokyo, Japan.
Hydrophilic interaction liquid chromatography (HILIC) offers unique advantages for the separation of very
polar compounds when compared to reversed-phase chromatography. A new silica based HILIC phase
was developed to provide additional selectivity options in HILIC separations. The separation of water
soluble vitamins on the new TSKgel NH2-100 HILIC column and on the well known TSKgel Amide-80
HILIC column demonstrates the differences in selectivity.
INTRODUCTION
SCHEMATIC DIAGRAM OF THE NEW HILIC PHASE
HILIC is used primarily to separate polar and hydrophilic compounds. Target applications for HILIC
include the analysis of saccharides, glycosides,
oligosaccharides, peptides and hydrophilic drugs.
Altering selectivity plays a major role in maximizing
resolution.
NH 2
CH 2
CH 2
R
H 3C
H 3C
HYDROPHILIC INTERACTION CHROMATOGRAPHY
HILIC PHASES
Typical HILIC stationary phases are silica or polymer particles carrying polar functional groups (e.g.,
amino, amide or zwitterionic groups). Conventional
amino type HILIC columns have limited stability in
aqueous solutions but sometimes the selectivity of
an amino ligand better suits the target application.
Therefore, we developed a new HILIC phase, which
combines the amino ligand functionality with high
durability.
The new amino type HILIC phase is based on a 3 µm
silica particle with 100 Å pores, which is treated with
a special endcapping procedure. Amino groups are
introduced step wisely after endcapping (Figure 1).
Si
CH 3
O
H 3C
Si
O O O
H 3C
Si
CH 3
O
Silica
Figure 2
The amino groups act as HILIC functional groups.
Because of a high ligand density and large surface
area TSKgel NH2-100 3 µm columns show the
strongest retention for polar compounds among
the commercially available HILIC columns.
H-U-PLOTS OF TSK-GEL AMIDE-80 AND NH-2-100 HILIC
30
NH2-100 3 µm
Amide-80 3 µm
25
HETP (µm)
HILIC has similarities to normal phase chromatography with regard to the nature of the stationary
phase. However, the eluents used for HILIC are
similar to those known from reversed phase chromatography, e.g mixtures of acetonitrile and water
or aqueous buffers, applied in isocratic or gradient
mode. Hydrogen bonding and dipole–dipole interactions are the dominating retention mechanisms
in HILIC mode. The number of polar groups, as well
as the conformation and solubility of the sample in
the mobile phase determine the elution order.
R:Spacer
20
15
10
5
0
0
5
10
15
Linear velocity (cm/min)
Figure 2
Conditions:
Columns:
TSKgel NH2-100 3 µm and TSKgel Amide-80
3 µm (4.6 mm ID x 15 cm L each)
Sample:
Uracil
Eluent:
H2O/acetonitrile = 10/90
Flow rates: 0.1∼2.4 mL/min
Temp.:
40 °C
Detection: UV @ 254 nm
      
m e e t s
i n n o v a t i o n !

i n s p i r a t i o n
#406
Figure 2 shows the H-u plots of the TSKgel Amide-80
and NH2-100 HILIC phases. The optimum HETP for the
amino type column is reached at a flow-rate of 1.2 mL/
min at a pressure of 6 MPa. At increased flow-rates the
H-u-curve is relatively flat. This allows using this column for fast separations at elevated flow rates without
impairing separation efficiency.
The availability of two TSK-GEL HILIC phases offering
different selectivity features allows for adopting both,
the stationary as well as the mobile phase to optimize
the separation of a given sample. Available in a particle size of 3 µm, both columns are ideally suited for
high efficiency HPLC as well as HPLC–MS analysis
SEPARATION OF WATER SOLBLE VITAMINS
MATERIAL AND METHODS
Columns: TSKgel NH2-100 3 µm, 4.6 mm ID × 15 cm L
TSKgel Amide-80 3 µm, 4.6 mm ID × 15 cm L
Mobile
phase:
25 mM phosphate buffer (pH 2.5)/ACN=30/70
Flow rate: 1 mL/min
Temp.:
40 °C
Detection: UV @ 254 nm
Sample: Figure 3: vitamin standard mixture
Figure 4: energy drink (filtrated, diluted in
CAN (1:1))
Injection: 5 µL
Figure 3 shows the separation of a standard solution
of water soluble vitamins on a TSKgel NH2-100 column compared to the separation on a TSKgel Amide80 column. Both columns have the same dimension
(4.6 mm ID × 15 cm L) and particle size (3 µm). Flow
rate and mobile phase were identical as well.
The elution order of the compounds varies when
applying the same mobile phase to both columns: The
new amino type column shows a stronger retention
for nicotinic acid, vitamin C and vitamin B12 while
retention of vitamin B1, B2 and pyridoxine is reduced.
Figure 4 shows the analysis of a commercially available energy drink on the new amino-type HILIC column, after filtration and addition of the same volume
of acetonitrile.
Figure 3
Columns:
Peaks:
TSKgel Amide-80 and TSKgel NH2-100
1 = nicotinamide; 2 = vitamin B2;
3 = pyridoxine; 4 = nicotinic acid;
5 = vitamin C; 6 = vitamin B1;
7 = vitamin B12
HILIC ANALYSIS OF WATER SOLUBLE VITAMINS
HILIC separations are performed either in isocratic
mode with a high percentage of organic solvent or
with gradients starting with high percentage of organic
solvent and ending with a high portion of aqueous solvent, which is opposite to reversed phase. The elution
order of compounds is usually inversed as well. As a
result in HILIC mode polar compounds are very well
separated according to increased polarity. We present
the analysis of water soluble vitamins (Nicotinamide,
Nicotinic acid, Pyridoxine and Vitamins B1, B2, B12
and C) as an example for the separation of polar compounds on the two available TSK-GEL HILIC phases.
For difficult separations of the very polar vitamins of
the B complex HILIC separation is advantageous over
reversed-phase analysis, especially when combined
with highly sensitive mass spectrometric detection.
i n s p i r a t i o n
m e e t s
i n n o v a t i o n
!
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



CONCLUSION
TSKgel Amide-80 HILIC columns have been used for
years for a broad range of HILIC applications. The new
3 µm TSKgel NH2-100 columns provide an additional

selectivity option, when
increased retention or alter


nate selectivity is 
needed. This new bonded phase

provides a powerful tool for robust method development in hydrophilic interaction liquid chromatography.
ANALYSIS OF VITAMINS IN AN ENERGY DRINK




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


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


 
Figure 4





 





 
















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